Several publications are referenced in this application by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Pluripotent stem cells give rise to all red blood cells, white blood cells and platelets. Stem cells are present in the circulation as well as in the bone marrow, though in smaller numbers. Ultimately, these cells leave the bloodstream and settle and proliferate in certain tissues, such as the spleen. This colonizing ability is important in the embryonic development of hematopoietic tissues. Blood cell formation occurs first in outlying extraembryonic regions of the mesoderm, then in the liver and spleen and finally in the bone marrow, where it persists throughout adult life.
The current paradigm concerning the kinetics of hematopoiesis is that only the most primitive pluripotential bone marrow (BM) stem cells can support prolonged hematopoiesis whereas more differentiated, lineage-committed stem cells can only contribute to a particular lineage for a limited period of time. All hematopoietic lineages, including the myeloid lineage, develop from pluripotential stem cells (PSC, also called primitive stem cells) which are found in bone marrow (BM) and spleen in the adult mouse (1). The accepted definition of a PSC is that it is capable of self-renewal and that it can differentiate into all lineages of the hematopoietic system (2–4). To date, a cell surface marker unique for PCS has not been found, nor do they respond specifically to any cytokine or growth factor. PCS are rare in BM (1 in 2,000–5,000 cells) and are contained within a population of BM cells with the phenotype Thy-1.110Lin-Sca-1+ (2,3,5–7). When this population is purified by negative selection with antibodies and complement followed by positive selection by FACS sorting, it contains all BM PSC (2,3,5–7). However, the PSC constitute only 10% (at best) of that population (6, 8). The rest of the cells in this population are a mix of various uncharacterized, more differentiated stem cells.
In the early 1960's Till and McCulloch classified BM stem cells as cells that form spleen nodules containing highly replicating hematopoietic stem cells, termed colony-forming-units-spleen (CFU-S), 8–12 days post reconstitution of irradiated recipients with adult BM cells (9). It was later realized that the observed spleen nodules contained myeloid and erythroid precursors only, and that their capacity to replenish the myeloid and erythroid lineages was transient at best. (10). More recent studies have shown that only the most primitive pluripotential BM stem cells can support hematopoiesis for long periods of time (2, 11–13). This observation is mostly based upon experiments in which the reconstitution potential of the PCS containing subpopulation (Thy-1.1lo LinSca-1+ cells) was compared to that of the remaining BM cells. In these experiments, only the PSC-enriched population could protect a lethally-irradiated host indefinitely (2, 6, 7).
Further support for this notion came from studies on the kinetics of hematopoiesis. Studies using retroviral-mediated tagging of BM-derived PSC with a reporter gene have shown that, for a short time immediately following the reconstitution of a lethally-irradiated mouse, many stem cell clones contribute to hematopoiesis. However, a few weeks later, only very few clones were contributing to the continuous replenishment of all hematopoietic lineages (14, 15). These results suggest that for a short time after reconstitution, both lineage-committed as well as PSC contribute to hematopoiesis but the more mature, lineage-committed cells are short-lived and therefore disappear within a few weeks. Similar conclusions were reached by Harrison et al. Using a competitive repopulation assay (16), these authors showed that the appearance of all 3 hematopoietic lineages is highly correlated with respect to time which they interpreted as an indication that most donor cells arise from the same PSC and therefore, lineage-committed precursors present in the inoculum could not have contributed significantly to any of the lineages.